This is a method where you take a bunch of protein crystals whose identity is uncertain and disolve them and run them on a gel. Do not use diffraction quality crystals for this unless you have a ton of them.
Step 1 - Select your crystals: very small crystals widely dispersed around the drop are extremely hard to handle and separate from the protein in the drop. I recommend using clustered groups of larger crystals which while being useless for data collection are perfectly servicable for this purpose. You need to use 2-3 clusters about 0.1mm across to get enough protein.
Step 2 - dilute the drop: do all manipulation on the microscope, and do it while watching the crystals. It is of little use if your crystals dissolve while you are trying to select them. Usually the well buffer works pretty well. Open up the drop and invert it on the microscope stage (drop upwards) and refocus the microscope until you can see the crystals at low power. Add 5-10 µl of well buffer to the drop.. If the crystals are on the surface of the drop, try pipetting the well buffer directly onto them to dislodge them (you want to put them into the body of the expanded drop). If they are on a skin on the surface of the drop, you will probably have to use the tools to separate them from the skin. This can be difficult with small crystals. Assuming your crystal is behaving itself, add well buffer to expand the volume of the drop, then suck out most of the liquid (leave the crystal in the drop) and discard it. Do this several times to dilute the protein within the drop as much as possible. If practical, directly pippette the well buffer onto the crystals to wash them as much as possible. If there are phase blobs or precipitate in the drop try and suck that up to remove as much non-crystalline protein from the drop as possible on each successive wash. Change tips often.
Step 3 - remove the crystal: take 50-100 µl of well buffer and put it in a shallow depression dish (e.g. the Pyrex 3-well plates). Using a P2 or P20 with a small trip, suck out the crystal from the expanded drop with as little liquid as possible and pipette it into the large volume of well buffer. Relocate the crystal in the new volume with the microscope. If possible wash the crystal directly with the well buffer. If you have enough well buffer you can do this several times, although if you wash the crystal probably once will suffice.
Step 4 - add to loading buffer: with the P2/P20, suck the well-washed crystal up and add it in as small a volume as possible to gel loading buffer. Discard all the other solutions. Repeat from step 1 until you've got enough crystals in the loading buffer.
Run the gel as normal, and silver-stain it for extra sensitivity. There will probably be some protein carry-over but the amount of contamination will be quite small unless the protein has crashed out onto the crystal.
Last modified: Feb 2006 by Phil Jeffrey